Simple catalytic DNA biosensors for ions based on color changes

ABSTRACT

Disclosed are compositions and methods for the sensitive and selective detection of ions using nucleic acid enzymes and DNA modified microparticles.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government may have rights in the present invention pursuant tothe terms of grant number DEFG02-01ER63179 awarded by the Department ofEnergy.

BACKGROUND

Many metals pose a risk as environmental contaminants. A well-knownexample is lead. Low level lead exposure can lead to a number of adversehealth effects, with as many as 9-25% of pre-school children presentlyat risk. The level of lead in the blood considered toxic is ≧10 μg/dL(480 nM). Current methods for lead analysis, such as atomic absorptionspectrometry, inductively coupled plasma mass spectrometry, and anodicstripping voltammetry, often require sophisticated equipment, samplepre-treatment, and skilled operators.

Simple, rapid, inexpensive, selective and sensitive methods that permitreal time detection of Pb²⁺ and other metal ions are very important infields such as environmental monitoring, clinical toxicology, wastewatertreatment, and industrial process monitoring. Furthermore, methods areneeded for monitoring free or bioavailable, instead of total, metal ionsin industrial and biological systems.

Many fluorescent chemosensors, including fluorophore-labeled organicchelators (Rurack, et al., 2000; Hennrich et al., 1999; Winkler et al.,1998; Oehme & Wolfbeis, 1997) and peptides (Walkup & Imperiali, 1996;Deo & Godwin, 2000; Pearce et al., 1998), have been developed for metalion detection. These ion sensors are usually composed of an ion-bindingmotif and a fluorophore. Metal detection using these fluorescentchemosensors relies on the modulation of the fluorescent properties ofthe fluorophore by the metal-binding event. Detection limits on thelevel of micromolar and even nanomolar concentrations have been achievedfor heavy metal ions including Zn²⁺, Cu²⁺, Hg²⁺, Cd²⁺ and Ag⁺. Thedesign and synthesis of a chemosensor that exhibits highly selective andsensitive binding of the metal ion of choice in aqueous solution isstill a big challenge, although the metal binding and the fluorescentmoieties of the sensor can be systematically varied to achieve desiredproperties. Although fluorescence spectroscopy is a technique wellsuited for detecting very small concentrations of analytes, afluorometer is required to generate and detect the emitted signal. Thus,the need for expensive instrumentation and complicated operationprocedures make this method impractical for applications such ashousehold use, field testing or small clinic testing.

Nucleic acid molecules have previously been adapted to sense thepresence of nucleic acids and to detect gene mutations from inheriteddiseases or chemical damages. In recent years, the molecular recognitionand catalytic function of nucleic acids have been extensively explored.This exploration has lead to the development of aptamers and nucleicacid enzymes.

Aptamers are single-stranded oligonucleotides derived from an in vitroevolution protocol called systematic evolution of ligands by exponentialenrichment (SELEX). Nucleic acid aptamers have been isolated from randomsequence pools and can selectively bind to non-nucleic acid targets,such as small organic molecules or proteins, with affinities as high as10⁻¹⁴ M (Uphoff et al., 1996; Famulok, 1999). Most aptamers undergo aconformational change when binding their cognate ligands. With thisproperty, several DNA and RNA aptamers have been engineered to senseL-adenosine or thrombin through an internally labeled fluorescentreporter group (Jhaveri et al., 2000). Here, the conformational changein the aptamer upon binding leads to a change in fluorescence.

Nucleic acid enzymes are nucleic acid molecules that catalyze a chemicalreaction. In vitro selection of nucleic acid enzymes from a library of10¹⁴-10¹⁵ random nucleic acid sequences offers considerable opportunityfor developing enzymes with desired characteristics (Breaker & Joyce,1994; Breaker, 1997). Compared with combinatorial searches of chemo- andpeptidyl-sensors, in vitro selection of DNA/RNA is capable of sampling alarger pool of sequences, amplifying the desired sequences by polymerasechain reactions (PCR), and introducing mutations to improve performanceby mutagenic PCR.

Allosteric ribozymes (or aptazymes), which combine the features of bothaptamer and catalytic RNA, also hold promises for sensing smallmolecules (Potyrailo et al., 1998; Koizumi et al., 1999; Robertson &Ellington, 1999, 2000). Their reactivity is modulated through theconformational changes caused by the binding of small organic moleculesto an allosteric aptamer domain. Therefore, the signal of ligand bindingcan be transformed into a signal related to chemical reaction.

Divalent metal ions can be considered as a special class of cofactorscontrolling the activity of nucleic acid enzymes. The reaction rate ofthe nucleic acid enzymes depends on the type and concentration of themetal ion in solution. Several RNA and DNA enzymes obtained through invitro selection are highly specific for Cu²⁺, Zn²⁺, and Pb²⁺, with metalion requirements on the level of micromolar concentrations (Breaker &Joyce, 1994; Pan & Uhlenbeck, 1992; Carmi et al., 1996; Pan et al.,1994; Cuenoud & Szotak, 1995; Li et al., 2000; Santoro et al., 2000).

A variety of methods have been developed for assembling metal andsemiconductor colloids into nanomaterials. These methods have focused onthe use of covalent linker molecules that possess functionalities atopposing ends with chemical affinities for the colloids of interest. Oneof the most successful approaches to date, (Brust et al., (1995)),involves the use of gold colloids and well-established thiol adsorptionchemistry (Bain & Whitesides, (1989); Dubois & Nuzzo (1992)). In thisapproach, linear alkanedithiols are used as the particle linkermolecules. The thiol groups at each end of the linker moleculecovalently attach themselves to the colloidal particles to formaggregate structures. The drawbacks of this method are that the processis difficult to control and the assemblies are formed irreversibly.Methods for systematically controlling the assembly process are neededif the materials properties of these structures are to be exploitedfully.

The potential utility of DNA for the preparation of biomaterials and innanofabrication methods has been recognized. Researchers have focused onusing the sequence-specific molecular recognition properties ofoligonucleotides to design impressive structures with well-definedgeometric shapes and sizes. Shekhtman et al., (1993); Shaw & Wang,(1993); Chen et al., (1989); Chen & Seeman, (1991); Smith and Feigon(1992); Wang et al., (1993); Chen et al., (1994); Marsh et al., (1995);Mirkin (1994); Wells (1988); Wang et al., (1991). However, the theory ofproducing DNA structures is well ahead of experimental confirmation.Seeman et al., New J. Chem., 17, 739-755 (1993).

Agglutation assays are well known for the detection of various analytes.The basic principle of an agglutination assay is the formation of clumps(agglutination or aggregation) of small particles coated with a bindingreagent when exposed to a multi-valent binding partner specific for thebinding reagent. Particles routinely used in agglutation assays include,for example, latex particles, erythrocytes (RBCs), or bacterial cells(often stained to make the clumps visible). Typically, a bindingreagent, such as an antibody, is attached to the particles. A samplethought to contain the analyte of interest is contacted with asuspension of such coated particles. If the analyte is present, crosslinking of the particles occurs due to bond formation between theantibodies on the particles and the analyte in the sample. Such bindingresults in the agglutation of the particles which can be detected eithervisually or with the aid of simple instrumentation. In an alternativeprotocol, an antigen is attached to the particles and the presence of anantibody specific for the antigen detected in the sample.

More recently, metal, semiconductor and magnetic particles have beenused in aggregation assays. For example, particles comprising metal(e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe,CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g.,ferromagnetite) colloidal materials have been described. Other particletypes include ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe,In₂ S₃, In₂ Se₃, Cd₃ P₂, Cd₃ As₂, InAs, and GaAs.

Mirkin et al. (U.S. Pat. No. 6,361,944) describes aggregation assays fordetecting a nucleic acid in a sample. This method involves incubating asample thought to contain a nucleic acid with particles havingoligonucleotides attached to the surface (particle-oligonucleotideconjugates). The oligonucleotides on each particle have anoligonucleotide complementary to one of the sequences of at least twoportions of the nucleic acid. Alternatively, at least two types ofparticles having oligonucleotides attached may be used. Theoligonucleotides on the first type of particles having a sequencecomplementary to one portion of the nucleic acid and theoligonucleotides on the second type of particles have a sequencecomplementary to a second portion of the sequence of the nucleic acid.The incubation takes place under conditions effective to allowhybridization of the oligonucleotides on the particles with the nucleicacid in the sample. Such a hybridization results in an aggregation ofthe particles. This produces a color change which may be detectedvisually or with simple instrumentation. For example, the aggregation ofgold particles results in a color change from red to purple (Mirkin(U.S. Pat. No. 6,361,944)).

Methods of detection based on observing such a color change with thenaked eye are cheap, fast, simple, robust (the reagents are stable) anddo not require specialized or expensive equipment. This makes suchmethods particularly suitable for use in applications such as thedetection of lead in paint or heavy metals in water.

BRIEF SUMMARY

The present invention provides a method of detecting the presence of anion in a sample. A new class of DNA enzyme-based biosensor for ions isprovided. This combines the high selectivity of DNA enzymes with theconvenience of particle aggregation-based detection. Such selectivityand convenience provides for semi-quantitative and quantitativedetection of ions over a concentration range of several orders ofmagnitude.

The sample may be any solution that may contain an ion (before or afterpre-treatment). The sample may contain an unknown concentration of anion and may contain other ions. For example, the sample may be paintthat is tested for lead content. The sample may be diluted yet stillremains a sample. The sample may be obtained from the naturalenvironment, such as a lake, pond, or ocean, an industrial environment,such as a pool or waste stream, a research lab, common household, or abiological environment, such as blood.

In one embodiment, the invention provides a nucleic acid enzyme, asubstrate and particles. The nucleic acid enzyme, a substrate andparticles are provided as, or form an aggregate. The method fordetection of the ion comprises contacting the aggregate with the ionwherein the nucleic acid enzyme causes cleavage of the substrate in thepresence of the ion. The method for detection of the ion may beperformed in the presence of other ions.

In a preferred embodiment, the particles are gold particles and thepresence of the ion in detected by observing a color change resultingfrom a breakdown of the aggregate.

In another aspect, the invention provides kits for the detection of anion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Selection scheme for RNA-cleaving deoxyribozymes. FIG. 1A. (SEQID NO: 12) Starting pool of random-sequenced DNAs, engineered to containtwo substrate-binding domains. Each member of the pool contains a5′-terminal biotin (encircled B), a single embedded ribonucleotide (rA)and a 40-nucleotide random sequence domain (N40). FIG. 1B. Selectiveamplification scheme for isolation of DNA that catalyzes the metalcofactor (Co²⁺ or Zn²⁺) dependent cleavage of an RNA phosphodiester.

FIG. 2. (SEQ ID NOS 13-23, respectively, in order of appearance)Sequence classes of the cloned Zn-DNA. The numbers on the left are theclone-numbers randomly assigned to the sequences during the cloning andsequencing process. The highly conserved sequences (Region-20nt) are inbold. The covariant nucleotides are underlined. The 5′- and the3′-primer binding sequences are shown in italic.

FIG. 3. (SEQ ID NOS 24-42, respectively, in order of appearance)Sequence classes of the cloned Co-DNA. The clone-numbers are listed onthe left. The 5′ and the 3′ primer binding sequences are in italic.

FIG. 4. (SEQ ID NOS 43-70, respectively, in order of appearance)Sequence alignment of the N40 region of the reselected Zn-DNAs. Thewild-type sequence is listed on the top, followed by the reselectedZn-DNA sequences. Only the point mutations are shown for the reselectedsequences, while the nucleotides that are identical to the wild type atthe corresponding positions are omitted. Numbers listed on the left areclone-numbers. The rate constants (k_(obs)) of several reselected Zn-DNAin 100 μM Zn²⁺ are shown on the right.

FIG. 5. (SEQ ID NOS 1 & 2) Proposed secondary structure of theZn(II)-dependent trans-cleaving deoxyribozyme.

FIG. 6. Sequences and proposed secondary-structures of severalRNA-cleaving deoxyribozymes. FIG. 6A (SEQ ID NOS 71 & 72) and FIG. 6B(SEQ ID NOS 73 & 74). The deoxyribozyme selected using Mg²⁺ or Pb²⁺ ascofactor (Breaker & Joyce, 1994, 1995). FIG. 6C (SEQ ID NOS 75 & 76) andFIG. 6D (SEQ ID NOS 77 & 78). The 1{tilde over (0)}23 and the {tildeover (8)}17 deoxyribozymes selected in Mg²⁺ to cleave all-RNA substrate(Santoro & Joyce, 1997). FIG. 6E (SEQ ID NOS 79 & 80). A deoxyribozymeselected using L-histidine as cofactor. FIG. 6F (SEQ ID NOS 81 & 82).The 17E deoxyribozyme selected in Zn²⁺. In each structure, the upperstrand is the substrate and the lower strand is the enzyme. Arrowsidentify the site of RNA transesterification.

FIG. 7. Comparison of G3 deoxyribozyme with class II Co-DNA. FIG. 7A.(SEQ ID NO: 83) The predicted secondary structure of the G3deoxyribozyme (Geyer & Sen, 1997). X represents variable sequences. Theboxed region was also found in class II Co-DNA. FIG. 7B. (SEQ ID NO: 84)The minimal structure motif of the class II Co-DNA predicted by mfoldprogram. The arrows indicate the cleavage sites.

FIG. 8. Figure showing an assay protocol for particle based DNA assayfor Pb⁺⁺.

DETAILED DESCRIPTION

The invention described herein represents a new class of DNAenzyme-based biosensor for ions. It combines the high selectivity of DNAenzymes with the convenience of particle aggregation-based detection.Such selectivity and convenience provides for semi-quantitative andquantitative detection of ions over a concentration range of severalorders of magnitude. In a preferred embodiment, the aggregation-baseddetection domain is decoupled from the ion-recognition/catalysis domain,and therefore the sensitivity and selectivity of this system may bemanipulated by a careful choice of particles and aggregation method, andby performing in vitro selection of ion-binding domains to not only keepsequences reactive with the ion of choice, but also remove sequencesthat also respond to other ions.

Nucleic Acid Enzymes

A growing number of nucleic acid enzymes have been discovered ordeveloped showing a great diversity in catalytic activity (Table 1 andTable 2). Many if not all of the enzymes are dependent on one or moreion cofactors. In vitro selection may be used to “enhance” selectivityand sensitivity for a particular ion. Such enzymes find particularutility in the compositions and methods of the present invention. Forexample, nucleic acid enzymes that catalyze molecular association(ligation, phophorylation, and amide bond formation) or dissociation(cleavage or transfer) are particularly useful.

In preferred embodiments, a nucleic acid enzyme that catalyzes thecleavage of a nucleic acid in the presence of an ion is used. Thenucleic acid enzyme may be RNA (ribozyme), DNA (deoxyribozyme), aDNA/RNA hybrid enzyme, or a peptide nucleic acid (PNA) enzyme. PNAscomprise a polyamide backbone and the bases found in naturally occurringnucleosides and are commercially available from, e.g., Biosearch, Inc.(Bedford, Mass.).

Ribozymes that may be used in the present invention include, but are notlimited to, group I and group II introns, the RNA component of thebacterial ribonuclease P, hammerhead, hairpin, hepatitis delta virus andNeurospora VS ribozymes. Also included are in vitro selected ribozymes,such as those isolated by Tang and Breaker (2000).

One limitation of using a ribozyme is that they tend to be less stablethan deoxyribozymes. Thus, in preferred embodiments, the nucleic acidenzyme is a deoxyribozyme. Preferred deoxyribozymes include those shownin FIGS. 6A-6F and deoxyribozymes with extended chemical functionality(Santoro et al., 2000).

TABLE 1 Reactions catalyzed by ribozymes that were isolated from invitro selection experiments. Reaction k_(cat) (min⁻¹) K_(m) (μM)k_(cat)/k_(uncat) ^(a) Reference Phosphoester centers Cleavage 0.1 0.0310⁵ Vaish, 1998 Transfer 0.3 0.02 10¹³ Tsang, 1996 Ligation 100 9 10⁹Ekland, 1995 Phosphorylation 0.3 40 >10⁵ Lorsch, 1994 Mononucleotide 0.35000 >10⁷ Ekland, 1996 polymerization Carbon centers Aminoacylation 19000 10⁶ Illangasekare, 1997 Aminoacyl ester 0.02 0.5 10 Piccirilli,1992 hydrolysis Aminoacyl transfer 0.2 0.05 10³ Lohse, 1996 N-alkylation0.6 1000 10⁷ Wilson, 1995 S-alkylation 4 × 10⁻³ 370 10³ Wecker, 1996Amide bond cleavage 1 × 10⁻⁵ 10² Dai, 1995 Amide bond formation 0.04 210⁵ Wiegand, 1997 Peptide bond formation 0.05 200 10⁶ Zhang, 1997Diels-Alder >0.1 >500 10³ Tarasow, 1997 cycloaddition Others Biphenylisomerization 3 × 10⁵ 500 10² Prudent, 1994 Porphyrin metallation 0.9 1010³ Conn, 1996 ^(a)Reactions catalyzed by ribozymes that were isolatedfrom in vitro selection experiments. kcat/kuncat is the rate enhancementover uncatalyzed reaction.

TABLE 2 Deoxyribozymes isolated through in vitro selection. k_(max)k_(cat)/ Reaction Cofactor (min⁻¹)^(a) k_(uncat) Reference RNA Pb²⁺ 110⁵ Breaker, 1994 transesterification Mg²⁺ 0.01 10⁵ Breaker, 1995 Ca²⁺0.08 10⁵ Faulhammer, 1997 Mg²⁺ 10 >10⁵ Santoro, 1997 None 0.01 10⁸Geyer, 1997 L-histidine 0.2 10⁶ Roth, 1998 Zn²⁺ ~40 >10⁵ Li, J., 2000DNA cleavage Cu²⁺ 0.2 >10⁶ Carmi, 1996 DNA ligation Cu²⁺ or Zn²⁺ 0.0710⁵ Cuenod, 1995 DNA phosphorylation Ca²⁺ 0.01 10⁹ Li, Y., 19995′,5′-pyrophophate Cu²⁺ 5 × 10⁻³ >10¹⁰ Li, Y., 2000 formation Porphyrinmetalation None 1.3 10³ Li, Y., 1996 ^(a)k_(max) is the maximal rateconstant obtained under optimized conditions.

An advantage of ribozymes and deoxyribozymes is that they may beproduced and reproduced using biological enzymes and appropriatetemplates. However, the present invention is not limited to ribozymesand deoxyribozymes. Nucleic acid enzymes that are produced by chemicaloligosynthesis methods are also included. Thus, nucleic acids includingnucleotides containing modified bases, phosphate, or sugars may be usedin the compositions and methods of the present invention. Modified basesare well known in the art and include inosine, nebularine, 2-aminopurineriboside, N⁷-denzaadenosine, and O⁶-methylguanosine (Earnshaw & Gait,1998). Modified sugars and phosphates are also well known and include2′-deoxynucleoside, abasic, propyl, phosphorothioate, and 2′-O-allylnucleoside (Earnshaw & Gait, 1998). DNA/RNA hybrids and PNAs may be usedin the compositions and methods of the present invention. The stabilityof PNAs and relative resistance to cellular nucleases make PNA enzymesamenable to in vivo applications.

In certain embodiments, the substrate for the nucleic acid enzyme andthe enzyme itself are contained in the same nucleic acid strand. Suchenzymes are cis-acting enzymes. Examples include the Zn²⁺-dependentdeoxyribozymes (Zn-DNA) created in Example 2 (FIG. 1A and FIG. 2).

In preferred embodiments, the nucleic acid enzyme cleaves a nucleic acidstrand that is separate from the strand comprising the enzyme(trans-acting). One advantage of utilizing trans-activity is that, aftercleavage, the product is removed and additional substrate may be cleavedby the enzymatic strand. A preferred nucleic acid enzyme is5′-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3′ (17E; FIG. 5; SEQ ID NO: 1). Thecorresponding preferred substrate to 17E is 5′-ACTCACTATrAGGAAGAGATG-3′(17DS; FIG. 5; SEQ ID NO: 2), where rA denotes a single ribonucleotide.

It may be beneficial to use directed mutation to change one or moreproperties of a nucleic acid enzyme or its substrate. Using 17E and 17DSas an example, one may wish to alter the avidity of the two arms of thehybridized enzyme and substrate. The “arms” are those areas displayingWatson-Crick base pairing in FIG. 5. To alter avidity, one may increaseor decrease the length of the arms. Increasing the length of the armsincreases the number of Watson-Crick bonds, thus increasing the avidity.The opposite is true for decreasing the length of the arms. Decreasingthe avidity of the arms facilitates the removal of substrate from theenzyme, thus allowing faster enzymatic turnover.

Another method of decreasing avidity includes creating mismatchesbetween the enzyme and the substrate. Alternatively, the G-C content ofthe arms may be altered. Of course, the effect of any directed changeshould be monitored to ensure that the enzyme retains its desiredactivity, including ion sensitivity and selectivity. In light of thepresent disclosure, one of skill in the art would understand how tomonitor for a desired enzymatic activity. For example, to ensure thatthe mutated enzyme maintained sensitivity and selectivity for Pb²⁺, onewould test to determine if the mutated enzyme remained reactive in thepresence of lead (sensitivity) and maintained its lower level ofactivity in the presence of other ions (selectivity).

The nucleic acid enzyme is sensitive and selective for a single ion. Theion may be any anion, for example, arsenate (AsO₄ ³⁻), or cation. Theion may be monovalent, divalent, trivalent, or polyvalent. Examples ofmonovalent cations include K⁺, Na⁺, Li⁺, Tl⁺, NH₄ ⁺ and Ag⁺. Examples ofdivalent cations include Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺,Pb²⁺, Hg²⁺, Pt²⁺, Ra²⁺, Ba²⁺, UO₂ ²⁺ and Sr²⁺. Examples of trivalentcations include Co³⁺, Cr³⁺, and lanthanide ions (Ln³⁺). Polyvalentcations include Ce⁴⁺, Cr⁶⁺, spermine, and spermidine. The ion detectedby the biosensor also includes ions having a metal in a variety ofoxidation states. Examples include K(I), Na(I), Li(I), Tl(I), Ag(I),Hg(I), Mg(II), Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Pb(II),Hg(II), Pt(II), Ra(II), Ba(II), Sr(II), Co(III), Cr(III), Ln(III),Ce(IV), Cr(VI) and U(VI).

The biosensors of the present invention may be used to monitorcontaminants in the environment; in such a case preferred ions are thosethat are toxic to living organisms, e.g. Ag⁺, Pb²⁺ and Hg²⁺.

Often the nucleic acid enzymes that have activity with one ion also haveat least some activity with one or more other ions. Such multi-sensitiveenzymes may still be used in the compositions and methods of the presentinvention. However, it should be understood that use of amulti-sensitive enzyme may lead to uncertainty as to which of the ionsis present. In such cases, measuring the rate of enzymatic activity,using serial dilutions, or using an array of nucleic acid enzymes may behelpful in deciphering which ion is present.

In Vitro Selection of Nucleic Acid Enzymes

Many nucleic acid enzymes that are dependent on ions, particularly metalions, for activity are known in the art (Breaker & Joyce, 1994; Pan &Uhlenbeck, 1992; Cuenoud & Szostak, 1995; Carmi et al., 1996; Li et al.,2000; Santoro et al., 2000). In light of the present disclosure, one ofskill in the art would understand how to utilize a known nucleic acidenzyme in the methods and biosensors of the present invention.Furthermore, the present invention may include a nucleic acid enzymecreated by in vitro selection. Methods of in vitro selection of nucleicacid enzymes are known in the art and described herein.

In vitro selection is a technique in which RNA or DNA molecules withcertain functions are isolated from a large number of sequence variantsthrough multiple cycles of selection and amplification (Joyce, 1994;Chapman et al., 1994). The concept of in vitro selection of catalyticRNA molecules was first introduced in the late 1980's. Since then, ithas been widely applied to obtain ribozymes with maximized activities ornovel catalytic abilities, and to identify oligonucleotides (calledaptamers) that bind to certain proteins or small molecules with highaffinity. The process for aptamers selection is sometimes referred assystematic evolution of ligands by exponential enrichment (SELEX)(Tuerk& Gold, 1990).

The first catalytic DNA (deoxyribozyme) was isolated by Breaker andJoyce in 1994 through in vitro selection. This deoxyribozyme is able tocatalyze phosphodiester cleavage reaction in the presence of Pb²⁺.Unlike RNA-based catalysts, DNA molecules with catalytic functions havenot been encountered in nature, where DNA exists primarily asbase-paired duplex and serves mainly as the carrier of geneticinformation. The identification of DNA molecules with catalyticfunctions further demonstrated the power of in vitro selection.

In vitro selection is typically initiated with a large collection ofrandomized sequences. A typical DNA or RNA library for selectioncontains 10¹³-10¹⁶ sequence variants. The construction of a completelyrandomized pool is accomplished by chemical synthesis of a set ofdegenerated oligonucleotides using standard phosphoramidite chemistry.The 3′-phosphoramidite compounds of four nucleosides (A, C, G, and T)are premixed before being supplied to an automated DNA synthesizer toproduce oligonucleotides. By controlling the ratio of fourphosphoroamidites, the identity at each nucleotide position can beeither completely random, i.e. with equal chance for each base, orbiased toward a single base. Other strategies for creating a randomizedDNA library include applying mutagenic polymerase chain reaction (PCR)and template-directed mutagenesis (Tsang and Joyce, 1996; Cadwell andJoyce, 1992, 1994). For the purpose of in vitro selection of functionalRNA molecules, the randomized DNA library is converted to an RNA librarythrough in vitro transcription.

In vitro selection takes advantage of a unique property of RNA and DNA,i.e., the same molecule can possess both genotype (coding information)and phenotype (encoded function). The DNA or RNA molecules in therandomized library are screened simultaneously. Those sequences thatexhibit a desired function (phenotype) are separated from the inactivemolecules. Usually the separation is performed through affinity columnchromatography, being linked to or released from a solid support, gelelectrophoresis separation, or selective amplification of a taggedreaction intermediate. The genotype of the active molecules are thencopied and amplified, normally through polymerase chain reaction (PCR)for DNA or isothermal amplification reaction for RNA. Mutations can beperformed with mutagenic PCR to reintroduce diversity to the evolvingsystem. These three steps—selection, amplification and mutation, arerepeated, often with increasing selection stringency, until sequenceswith the desired activity dominate the pool.

Novel nucleic acid enzymes isolated from random sequences in vitro haveextended the catalytic repertoire of RNA and DNA far beyond what hasbeen found in nature. The selected ribozymes are capable of catalyzing awide range of reactions at both phosphate and non-phosphate centers(Table 1). The reactions that are catalyzed by deoxyribozymes are lessdiverse, compared with the ribozymes (Table 2). However, the catalyticrate (k_(cat)) of most deoxyribozymes is comparable to that of theribozymes catalyzing the same reaction. In certain cases, the catalyticefficiency (k_(cat)/K_(m)) of nucleic acid enzymes even exceeds that ofthe protein enzymes.

In vitro selection can be used to change the ion specificity or bindingaffinity of existing ribozymes, or to obtain nucleic acid enzymesspecific for desired ions. For example, in vitro-selected variants ofthe group I intron (Lehman & Joyce, 1993) and the RNase P ribozyme(Frank & Pace, 1997) have greatly improved activity in Ca²⁺, which isnot an active metal ion cofactor for native ribozymes. The Mg²⁺concentration required for optimal hammerhead ribozyme activity has beenlowered using in vitro selection to improve the enzyme performance underphysiological conditions (Conaty et al., 1999; Zillman et al., 1997).Breaker and Joyce have isolated several RNA-cleaving deoxyribozymesusing Mg²⁺, Mn²⁺, Zn²⁺, or Pb²⁺ as the cofactor (Breaker & Joyce, 1994,1995). Only the sequence and structure of the Pb²⁺-dependent and theMg²⁺-dependent deoxyribozymes were reported (FIG. 6A and 6B). Otherexamples of metal-specific RNA/DNA enzymes obtained through in vitroselection include a Pb²⁺-specific RNA-cleaving ribozyme (calledleadzyme)(Pan & Uhlenbeck, 1992), a Cu²⁺-specific DNA-cleavingdeoxyribozyme (Carmi et al., 1996), and a DNA ligase active in Zn²⁺ andCu²⁺ (Cuonod & Szostak, 1995).

Often nucleic acid enzymes developed for a specific metal ion by invitro selection will have activity in the presence of other metal ions.For example, 17E deoxyribozyme was developed by in vitro selection foractivity in the presence of Zn²⁺. Surprisingly, the enzyme showedgreater activity in the presence of Pb²⁺ than Zn²⁺. Thus, althoughproduced in a process looking for Zn²⁺-related activity, 17E may be usedas a sensitive and selective sensor of Pb²⁺.

To produce nucleic acid enzymes with greater selectivity, a negativeselection step may be included in the process. For Example,Pb²⁺-specific deoxyribozymes may be isolated using a similar selectionscheme as for the selection of Co²⁺- and Zn²⁺-dependent DNA enzymesdescribed in Example 2. In order to obtain deoxyribozymes with highspecificity for Pb²⁺, negative-selections may be carried out in additionto the positive selections in the presence of Pb²⁺.

For negative selection, the DNA pool is selected against a “metal soup”,which contains various divalent metal ions (e.g. Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺,Cd²⁺, Co²⁺, Cu²⁺, etc.). Those sequences that undergo self-cleavage inthe presence of divalent metal ions other than Pb²⁺ are then washed offthe column. The remaining sequences are further selected with Pb²⁺ asthe cofactor. Pb²⁺-dependent deoxyribozymes with different affinitiesfor Pb²⁺ can be obtained by controlling the reaction stringency (Pb²⁺concentration). Besides the oligonucleotide sequences discussed here,other sequences shall be useful in the practicing of this invention.Such sequences are described in U.S patent application Ser. No.09/605558, filed Jun. 27, 2000, the contents of which are incorporatedby this reference.

DNA Coated Particles

Methods of making metal, semiconductor and magnetic particles arewell-known in the art. Methods of making ZnS, ZnO, TiO₂, AgI, AgBr,HgI₂, PbS, PbSe, ZnTe, CdTe, In₂ S₃, In₂ Se₃, Cd₃ P₂, Cd₃ As₂, InAs, andGaAs particles are also known. See, e.g., Mirkin et al. U.S. Pat. No.6,361,944. Suitable particles are also commercially available from,e.g., Amersham Biosciences, (Piscataway, N.J.) (gold) and Nanoprobes,Inc. (Yaphank, N.Y.) (gold).

In addition, other types of particles may be used in the presentinvention. For example, polystyrene latex particles or latex particlescontaining dye may be used. The critical requirement is that adetectable change must occur upon aggregation/aggregate breakdown. Inaddition, the composition of the particles must be such that theparticles do not interfere with the cleavage of the substrate by thenucleic acid enzyme in the presence of the metal ion.

Gold colloidal particles are preferred for use in detecting metal ions.Gold colloidal particles have high extinction coefficients for the bandsthat give rise to their intense colors. These colors vary with particlesize, concentration, interparticle distance, and extent of aggregationand shape of the aggregates, making these materials particularly usefulfor calorimetric assays. For instance, hybridization of oligonucleotidesattached to gold particles with oligonucleotides and nucleic acidsresults in an immediate color change visible to the naked eye (see,e.g., Mirkin et al. U.S. Pat. No. 6,361,944).

Gold particles, oligonucleotides or both are functionalized in order toattach the oligonucleotides to the particles. Such methods are known inthe art. For instance, oligonucleotides functionalized with alkanethiolsat their 3′-termini or 5′-termini readily attach to gold particles. SeeWhitesides (1995). See also, Mucic et al. (1996) (describes a method ofattaching 3′ thiol DNA to flat gold surfaces; this method can be used toattach oligonucleotides to particles). The alkanethiol method can alsobe used to attach oligonucleotides to other metal, semiconductor andmagnetic colloids and to the other particles listed above. Otherfunctional groups for attaching oligonucleotides to solid surfacesinclude phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 forthe binding of oligonucleotide-phosphorothioates to gold surfaces),substituted alkylsiloxanes (see, e.g. Burwell (1974) and Matteucci andCaruthers (1981) for binding of oligonucleotides to silica and glasssurfaces, and Grabar et al., (1995) for binding of aminoalkylsiloxanesand for similar binding of mercaptoaklylsiloxanes). Oligonucleotidesterminated with a 5′ thionucleoside or a 3′ thionucleoside may also beused for attaching oligonucleotides to solid surfaces. Gold particlesmay be attached to oligonucleotides using biotin-labeledoligonucleotides and streptavidin-gold conjugate colloids; thebiotin-streptavidin interaction attaches the colloids to theoligonucleotide. Shaiu et al., (1993). The following references describeother methods which may be employed to attached oligonucleotides toparticles: Nuzzo et al., (1987) (disulfides on gold); Allara and Nuzzo(1985) (carboxylic acids on aluminum); Allara and Tompkins (1974)(carboxylic acids on copper); Iler, (1979) (carboxylic acids on silica);Timmons and Zisman, (1965) (carboxylic acids on platinum); Soriaga andHubbard, (1982) (aromatic ring compounds on platinum); Maoz and Sagiv,(1987) (silanes on silica).

Each particle will have a plurality of oligonucleotides attached to it.As a result, each particle-oligonucleotide conjugate can bind to aplurality of oligonucleotides or nucleic acids having the complementarysequence.

Oligonucleotides of defined sequences are used for a variety of purposesin the practice of the invention. Methods of making oligonucleotides ofa predetermined sequence are well-known. See, e.g., Sambrook et al.,(1989) and F. Eckstein (1991). Solid-phase synthesis methods arepreferred for both oligoribonucleotides and oligodeoxyribonucleotides(the well-known methods of synthesizing DNA are also useful forsynthesizing RNA). Oligoribonucleotides and oligodeoxyribonucleotidescan also be prepared enzymatically.

Biosensors

Described herein are nucleic acid enzymes that are dependent on thepresence of a specific ion for activity. Using particle labeling, it ispossible to measure enzymatic activity, and hence ion concentration,without the need of complex instrumentation. These qualities make thecompositions of the present invention excellent for use in biosensorsfor detecting ions.

Many biosensors utilizing nucleic acids are known in the art. Forexample, biosensors using aptamers have been developed for detectingmolecules such as thrombin or adenosine (Potyrailo et al., 1999; Lee &Walt, 2000). In light of the present disclosure, one of ordinary skillin the art would know how to modify the nucleic acid biosensors toinclude nucleic acid enzymes.

In a simple embodiment, a biosensor of the present invention comprises anucleic acid enzyme, a cleavable substrate for the nucleic acid enzyme,and particles labeled with oligoribonucleotides complementary to atleast two portions of the cleavable substrate. Preferably, the substrateis modified, for example, by extension of the 3′ and 5′ ends by a numberof bases which act as “sticky end” for annealing to the complementaryDNA strand on the particles. Modification of the substrate allowscomplexes comprising of substrate linked particles to be formed withoutinhibiting the nucleic acid enzyme/cleavable substrate interaction.However, where the substrate contains regions not critical forinteraction with the nucleic acid enzyme, modification may not benecessary.

In a method using this embodiment the nucleic acid enzyme, cleavablesubstrate, and labeled particles are combined to form aggregatedcomplexes. Such aggregates are contacted with a sample suspected ofcontaining an ion to which the enzyme is sensitive. In the presence ofthe ion, the enzyme cleaves the substrate causing a break-up of theaggregates. An example of such an assay protocol is shown in FIG. 8. Insome embodiments, heating of the aggregates to a temperature above the“melting point” of the complex may be necessary. In such an embodiment,the presence of the ion allows the enzyme to cleave the substrate,preventing “re-aggregation” of the complex. However, in certainembodiments, heating is not required (see, for example, Example 8).

A change in the aggregation state of the particles may be detected byobserving an color change which accompanies the change in state. Forexample, the a breakdown of aggregation of gold particles results in acolor change from purple to red. Furthermore, the amount of substratecleavage depends on the concentration of the ion. A low concentration ofion results in only partial cleavage of the substrate, producing amixture of single particles and aggregates with different degree ofaggregations. Hence, this simple color test can be used as asemi-qualitative or qualitative way to detect an ion, for example, Pb²⁺.The color difference can be amplified to improve the sensitivity of themethod. For example, this may be achieved by spotting the resultingsolution onto an alumina TLC plate. Different degrees of color changeare produced, depending on the amount of ion present. Alternatively, aquantitative assay can be achieved by measuring the optical spectra ofthe assay mixture.

The detectable change that occurs upon a change in the aggregation stateof the particles may be a color change, the formation of aggregates ofthe particles, or the precipitation of the aggregated particles. Thecolor changes can be observed with the naked eye or spectroscopically.The formation of aggregates of the particles can be observed by electronmicroscopy or by nephelometry. The precipitation of the aggregatedparticles can be observed with the naked eye or microscopically.However, changes observable with the naked eye are preferred,particularly a color change observable with the naked eye.

The observation of a color change with the naked eye can be made morereadily against a background of a contrasting color. For instance, whengold particles are used, the observation of a color change isfacilitated by spotting a sample of the hybridization solution on asolid white surface (such as silica or alumina TLC plates, filter paper,cellulose nitrate membranes, and nylon membranes) and allowing the spotto dry. Initially, the spot retains the color of the hybridizationsolution (which ranges from pink/red, in the absence of aggregation, topurplish-red/purple, if there has been aggregation). On drying, a bluespot develops if aggregation is present prior to spotting. In theabsence of aggregation (e.g., because the target ion is present), thespot is pink. The blue and the pink spots are stable and do not changeon subsequent cooling or heating or over time. They provide a convenientpermanent record of the test. No other steps are necessary to observethe color change.

Alternatively, assay results may be visualized by spotting a sample ontoa glass fiber filter (e.g., Borosilicate Microfiber Filter, 0.7 micronpore size, grade FG75, for use with gold particles 13 nm in size), whiledrawing the liquid through the filter. Subsequent rinsing with waterwashes the excess, non-hybridized probes through the filter, leavingbehind an observable spot comprising the aggregates. Additional methodsuseful in visualizing assay results are described in Mirkin et al. U.S.Pat. No. 6,361,944.

The above methods allow the ion to be detected in a variety or sampletypes, including a bodily fluids, and in the presence of other ions.Various techniques may be employed to improve the accuracy and precisionof the above method of observation. Standards containing known amountsof ion may be assayed along side the unknown sample and the colorchanges compared. Alternatively, standard color charts, similar to thoseused with pH papers, may be provided.

Many variants of these simple embodiments are included within the scopeof the invention. The specificity of the nucleic acid enzyme may bevaried to allow specific detection of a wide range of ions. In addition,the composition, size and surface properties of the particles may beoptimized to obtain preferred aggregation and visual properties.Similarly, parameters such as the particle/oligonucleotide probecoupling technology, and the length and sequence of the oligonucleotideprobes and substrate may be varied to optimize the performance of theassay.

The invention also provides kits for detecting ions. In one embodiment,the kit comprises at least one container, the container holding at leastone type of particles having oligonucleotides attached thereto; acleavable substrate; and a nucleic acid enzyme. The oligonucleotides onthe particles have a sequence complementary to the sequence of at leasta first and a second portion of the cleavable substrate. The first andsecond portions of the cleavable substrate are separated by a thirdportion of the substrate that is cleaved by the nucleic acid enzyme inthe presence of the ion. The above components may be supplied in anaggregated state.

In a second embodiment, the above kit comprises at least two types ofparticles having oligonucleotides attached thereto. A first type ofparticles has oligonucleotides which have a sequence complementary tothe sequence of a first portion of the cleavable substrate. A secondtype of particles has oligonucleotides which have a sequencecomplementary to the sequence of a second portion of the cleavablesubstrate. The first and second portions of the cleavable substrate areseparated by a third portion of the substrate that is cleaved by thenucleic acid enzyme in the presence of the ion.

When a kit is supplied, the different components of the composition maybe packaged in separate containers and admixed immediately before use.Such packaging of the components separately may permit long-term storageof the active components. For example, one of more of the particleshaving oligonucleotides attached thereto; the cleavable substrate; andthe nucleic acid enzyme are supplied in separate containers.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain one of more of the abovereagents, or buffers that have been packaged under a neutral,non-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers, such as polycarbonate,polystyrene, etc.; ceramic, metal or any other material typicallyemployed to hold similar reagents. Other examples of suitable containersinclude simple bottles that may be fabricated from similar substances asampules, and envelopes, that may comprise foil-lined interiors, such asaluminum or an alloy. Other containers include test tubes, vials,flasks, bottles, syringes, or the like. Containers may have a sterileaccess port, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to be mixed. Removable membranes may be glass,plastic, rubber, etc.

The kits may also contain other reagents and items useful for detectingions. The reagents may standard solutions containing known quantities ofthe ion, dilution and other buffers, pretreatment reagents, etc. Otheritems which may be provided as part of the kit include a solid surface(for visualizing aggregate break down) such as a TLC silica plate,microporous materials, syringes, pipettes, cuvettes and containers.Standard charts indicating the appearance of the particles in variousaggregation states, corresponding to the presence of different amountsof the ion under test, may be provided.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD−ROM, Zipdisc, videotape, audiotape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail.

EXAMPLES

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtainlike or similar results without departing from the spirit and scope ofthe invention.

Example 1 Preparation of Gold Particles

Gold colloids (13 nm diameter) were prepared by reduction of HAuCl₄ withcitrate as described in Mirkin, U.S. Pat. No. 6,361,944, Frens, (1973)and Grabar, (1995). Briefly, all glassware was cleaned in aqua regia (3parts HCl, 1 part HNO₃), rinsed with Nanopure H₂O, then oven dried priorto use. HAuCl₄ and sodium citrate were purchased from Aldrich ChemicalCompany. Aqueous HAuCl₄ (1 mM, 500 mL) was brought to reflux whilestirring and 38.8 mM sodium citrate (50 mL) added quickly. The solutioncolor changed from pale yellow to burgundy. Refluxing was continued for15 min. After cooling to room temperature, the red solution was filteredthrough a Micron Separations Inc. 1 micron filter. Gold colloids werecharacterized by UV-vis spectroscopy using a Hewlett Packard 8452A diodearray spectrophotometer and by Transmission Electron Microscopy (TEM)using a JEOL 2010 transmission electron microscope.

Example 2 In-vitro Selection of an Ion-dependent Deoxyribozyme

A partially random DNA library was used to obtain deoxyribozymes thatcleave RNA in the presence of Zn²⁺ or Co²⁺.

Oligonucleotides

DNA oligonucleotides were purchased from Integrated DNA TechnologiesInc., Coralville, Iowa. Sequences of the random DNA template and theprimers (P1, P2 and P3) used in PCR amplifications are listed below:

P1: (SEQ ID NO:3) 5′-GTGCCAAGCTTACCG-3′ P2: (SEQ ID NO:4)5′-CTGCAGAATTCTAATACGACTCACTATAGGAAGAGATGGCGAC-3′ P3: (SEQ ID NO:5)5′-GGGACGAATTCTAATACGACTCACTATrA-3′ Template for random DNA pool: (SEQID NO:6) 5′-GTGCCAAGCTTACCGTCAC-N40-GAGATCTCGCCATCTCTTCCTATAGTGAGTCGTATTAG-3′

Primer P1b and P3b are the 5′-biotinylated version of primers P1 and P3.Primer P1a and P3a were prepared by 5′-labeling P1 and P3 with [□-³²P]ATP (Amersham) and T4 polynucleotide kinase (Gibco). The DNA/RNAchimeric substrate (17DS) for trans-cleavage assays has the sequence5′-ACTCACTATrAGGAAGAGATG-3′ (SEQ ID NO: 2), where rA denotes a singleribonucleotide. The all-RNA substrate (17RS) with the same sequence waspurchased from Dharmacon Research Inc. The trans-cleaving deoxyribozyme17E has the sequence 5′-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3′ (SEQ IDNO:1). The deoxyribozyme named 17E1 is a variant of 17E with thesequence 5′-CATCTCTTTTGTCAGCGACTCGAAATAGTGAGT-3′ (SEQ ID NO:7). Alloligonucleotides were purified using denaturing polyacrylamide gelelectrophoresis and desalted with the SepPak nucleic acid purificationcartridges (Waters) before use.

Preparation of Random DNA Pool

The initial pool for DNA selection was prepared by template-directedextension followed by PCR amplification. The extension was carried outwith 200 pmol of DNA template containing a 40-nucleotide random sequenceregion, and 400 pmol of primer P3b in 20×100 μl reaction mixtures forfour thermal-cycles (1 min at 92° C., 1 min at 52° C., and 1 min at 72°C.). Reaction buffer also included 0.05 U/μl Taq polymerase (Gibco), 1.5mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25° C.), 0.01% gelatinand 0.2 mM of each dNTP. Subsequently, 1 nmol each of P1 and P3b wereadded to the extension product to allow four more cycles of PCRamplification. The products were precipitated with ethanol and dissolvedin 0.5 mL of buffer A, which contains 50 mM HEPES (pH 7.0), 500 mM (forZn-DNA selection) or 1 M (for Co-DNA selection) NaCl. About 20 μM EDTAwas also added to the buffer to chelate trace amount of divalent metalion contaminants.

In Vitro Selection

The random DNA pool was immobilized on a NeutrAvidin column (Pierce) byincubating with the column materials for 30 minutes. The mixture wasgently vortex-mixed a few times during the incubation. The unbound DNAstrands were eluted with at least 5×100 μl of buffer A. Thenon-biotinylated strands of immobilized DNA were washed off the columnwith 5×100 μl of freshly prepared 0.2 M NaOH and 20 μM EDTA. The columnwas then neutralized with 5×100 μl of buffer A. The cleavage reactionwas carried out by incubating the immobilized single-stranded DNAcontaining the single ribonucleotide (rA) with 3×20 μl of reactionbuffer (buffer A plus 1 mM ZnCl₂ or CoCl₂) over 1 h. The eluted DNAmolecules were pooled and precipitated with ethanol. A fraction of theselected DNA was amplified in 100 μl PCR reaction with 40 pmol each ofprimers P1 and P2 over 1{tilde over (0)}20 thermal cycles. One tenth ofthe PCR product was further amplified for six cycles with 50 pmol ofprimers P1 and P3b. The final PCR product was ethanol precipitated andused to initiate the next round of selection. During the selection ofZn(II)-dependent deoxyribozymes (called Zn-DNA hereafter), theconcentration of ZnCl₂ was kept constant at 100 μM in the reactionbuffer for the following rounds of selection. Reaction time wasgradually decreased from 1 h to 30 s within 12 rounds of selection. Forthe selection of Co(II)-dependent deoxyribozymes (called Co-DNAhereafter), the concentration of CoCl₂ was gradually decreased from 1 mMto 100 μM and the reaction time from 1 h to 1 min within 10 rounds ofselection. The twelfth generation of selectively amplified Zn-DNA andthe tenth generation of Co-DNA were cloned using TÃTOPO Cloning Kit(Invitrogen) and sequenced with T7 Sequenase 2.0 Quick-denatured PlasmidSequencing Kit (Amersham).

Reselection

Based on the sequence of class I Zn-DNA or Co-DNA, partially degenerateDNA template libraries for reselection were synthesized (Integrated DNATechnology Inc., Coralville, Iowa) with 20% degeneracy at the N40region. In other words, during the oligonucleotide synthesis of the N40region, the wild type sequence was introduced at a probability of 80% ateach position, while the other three nucleotides each occurred at aprobability of 6.67%. The reselection pool was prepared with 10 pmol oftemplate and 100 pmol of primers P1 and P3b using the same protocolpreviously described. With 10 pmol (number of molecules S=6×10¹²) ofpartially randomized template, the statistic parameters of the DNAlibrary used for reselection were calculated based on the followingequations.P(k,n,d)=[n!/(ñk)!k!]d ^(k)({tilde over (1)}d)^(ñk)  (1)N(k)=[n!/(ñk)!k!]3^(k)  (2)C(n,k)=SP(k,n,d)/N(k)  (3)

P(k,n,d) is the probability of having k mutations within n (number ofrandomized positions, n=40) nucleotide positions that have beenrandomized at a degeneracy of d. N(k) is the number of distinctsequences that have k mutations with respect to the prototype sequence.C(n,k) is the number of copies for each sequence that has k mutations.The reselection pool was expected to contain the wild type sequence, allpossible sequences with {tilde over (1)}8 point mutations, and asampling of the sequences with >8 point mutations. More than half of thepopulation contains ≧8 point-mutations. The protocol for reselection wasthe same as the primary selection, except that the reaction time wasdecreased from 20 min to 1 min and the concentration of ZnCl₂ or CoCl₂was decreased from 20 μM to 5 μM over six generations. The sixthgeneration of reselected Zn- or Co-DNA were cloned and sequenced aspreviously described.

Kinetic Assays of the Reselected Cis-Cleaving DNA

The 5′³²P-labeled precursor DNA for cis-cleavage assay was prepared byPCR-amplification of the selected DNA population or the cloned DNAplasmid with primer 1b and 3a. The double-stranded product wasimmobilized on a NeutrAvidin column through the biotin moiety on primerP1b. The catalytic strand of DNA was eluted off the column with 3×20 μlfreshly prepared 0.2 N NaOH and neutralized with 8 μl of 3 M sodiumacetate (pH 5.3) in the presence of 50 μg/ml bovine serum albumin(Sigma). Following ethanol precipitation, the single-stranded DNA waspurified on an 8% denaturing polyacrylamide gel and desalted with SepPaknucleic acid purification cartridge. Bovine serum albumin (50 μg/ml) wasadded to the gel-soaking buffer (0.2 M NaCl, 20 μM EDTA, 10 mM TRi{tildeover (s)}HCl, pH 7.5) to prevent the DNA from adhering to the tube. Theconcentration of the DNA was determined by scintillation counting theradioactivity.

The precursor DNA was dissolved in buffer A and incubated at roomtemperature for 10 min before CoCl₂ or ZnCl₂ was added. The reaction wasstopped with 50 mM EDTA, 90% formamide and 0.02% bromophenol blue.Reaction products were separated on an 8% denaturing polyacrylamide geland quantified with a Molecular Dynamic phosphorimager.

In Vitro Selection of Zn(II)- or Co(II)-Dependent Deoxyribozymes

The DNA molecules capable of cleaving an RNA bond in the presence ofCo²⁺ or Zn²⁺ were obtained through in vitro selection. The initial DNAlibrary for selection contains ˜10¹⁴ out of the possible 10²⁴ (=4⁴⁰) DNAsequences. These molecules consist of a random sequence domain of 40nucleotides flanked by two conserved primer-binding regions. Thesequence of the conserved region was designed in such a way that theycould form two potential substrate-binding regions (FIG. 1A). Aribonucleic adenosine was embedded in the 5′-conserved sequence regionand was intended to be the cleavage site, since an RNA bond is moresusceptible than a DNA bond toward hydrolytic cleavage. The intrinsichalf-life of the phosphodiester linkage in RNA at pH 7 and 25° C. isestimated to be 1,000 years. The corresponding value for DNA is 200million years.

The DNA pool was immobilized on a NeutrAvidin column through the biotinmoiety on the 5′ terminus of the DNA. Biotin and Avidin bind stronglywith an association constant of K_(a)=10¹⁵ M⁻¹. The sequences thatunderwent self-cleavage in the presence of Co²⁺ or Zn²⁺ were eluted offthe column, amplified and used to seed the next round of selection (FIG.1B). The selection stringency was increased during the selection processwith shorter reaction time and less available divalent metal ions. Theactivity of the selected Zn-DNA gradually increased until the twelfthgeneration and declined thereafter, while the highest activity wasachieved with the tenth generation of Co-DNA. Therefore the twelfthgeneration of Zn-DNA and the tenth generation of Co-DNA were cloned andsequenced. The cloned sequences can be divided into different classesbased on sequence similarity (FIG. 2 and FIG. 3).

Individual sequences of the cloned Zn-DNA and Co-DNA were randomlychosen and sampled for activity. Under the selection conditions (100 μMZn²⁺, 500 mM NaCl, 50 mM HEPES, pH 7.0, 25° C.), the observed rateconstants of Zn-DNAs from sequence-classes I and II were 0. 10.2 min⁻¹,while class III sequences were less active, with k_(obs) around 0.02min⁻¹. The cleavage rate of the initial pool was 2×10⁻⁷ min⁻¹.Therefore, a 10⁻⁵10⁶ fold increase in cleavage rate has been achieve forZn-DNA selection. The cleavage rates of all the randomly picked Co-DNAsequences were <0.02 min⁻¹ under the conditions for Co-DNA selections(100 μM Co²⁺, 1 M NaCl, 50 mM HEPES, pH 7.0, 25° C.). Interestingly,even in the buffer (1 M NaCl, 50 mM HEPES, pH 7.0) alone, the class IICo-DNA exhibited similar activity as in the presence of 100 μM Co²⁺ orZn²⁺.

Clone #5 of Zn-DNA (Zn-5) and clone #18 of Co-DNA (Co-18) showedrelatively high activity, as well as high frequency of occurrence,within their lineages. The k_(obs) were 0.17 min⁻¹ for Zn-5 (in 100 μMZn²⁺) and 0.02 min⁻ for Co-18 (in 100 μM Co²⁺). The sequences of Zn-5and Co-18 were partially randomized (see Material and Methods fordetails) and subjected to reselection in order to further improve thereactivity and metal-binding affinity, and to explore the sequencerequirement of the conserved catalytic motif. Based on equations(1){tilde over ( )}(3), the reselection pool was expected to contain thewild type sequence, all possible sequences with {tilde over (1)}8 pointmutations, and a sampling of the sequences with >8 point mutations. Morethan half of the population should contain ≧8 point mutations. Sixrounds of reselection were carried out with {tilde over (5)}20 μM Zn²⁺or Co²⁺, however the activity of the reselected DNA was similar to theactivity of the wild type sequences. Sequencing of the Zn-DNA from boththe initial selection and reselection revealed a highly conservedsequence region. Therefore the lack of activity improvement afterreselection likely reflects a sequence pool dominated by a few highlyreactive sequences.

Sequence Alignment and Structure Analysis of Zn-DNA

The sequences of thirty individual clones of initially selected Zn-DNAcan be divided into three major classes based on sequence similarity.Differences among members of each class were limited to a few pointmutations (FIG. 2). A highly conserved sequence region of 20 nt,5′-TX₁X₂X₃AGCY₁Y₂Y₃TCGAAATAGT-3′ (SEQ ID NO:8) (Region-20 nt), wasobserved in all but one sequence albeit at different locations. Thesequences of 5′-X₁X₂X₃-3′ and 3′-Y₃Y₂Y₁-5′ are complimentary andcovariant, indicating that they form base pair with each other:5′-X₁X₂X₃-3′3′-Y₃Y₂Y₁-5′

The secondary structures of the sequenced Zn-DNA were predicted usingZuker's DNA mfold program (seehttp://mfold.wustl.edu/˜folder/dna/form1.cgi) through minimization offolding energy. The most stable structures predicted for thosecontaining Region-20nt all contained a similar structure motif. Thiscommon motif consists of a pistol-shaped three-way helical junctionformed by a 3 bp hairpin, an 8 bp hairpin and a double helix linking tothe rest of the molecule. The 3 bp hairpin and its adjacentsingle-stranded regions are part of the Region-20nt. The ribonucleicadenosine is unpaired and positioned opposite of the 3 bp hairpin.

After reselection, twenty-eight Zn-DNA clones were sequenced (FIG. 4).When compared with the parental wild type sequence (class I Zn-DNA), thereselected Zn-DNA contained point mutations mostly outside ofRegion-20nt. About one third of these sequences have a T→A mutation atposition 73, converting the {tilde over (T)}T mismatch in the wild typesequence to a WatsoñCrick base pair. In one fourth of the reselectedDNAs, the 5 nucleotide single-stranded bulge of the three-way junctionhas the sequence 5′-ACGAA-3′, corresponding to 5′-TCGAA-3′ in the wildtype. Clone #17 (named ZnR17) of the reselected Zn-DNA is most activeunder selection conditions (FIG. 4). Structural analysis of ZnR17revealed two completed base-paired helices in the three-way junction.Therefore, it was engineered into a trans-cleaving deoxyribozyme bydeleting the sequences outside of the three-way junction and the loop ofthe 8 bp hairpin. Such truncation resulted in two individual stands,which hybridize with each other through two 9-10 bp helices. The strandcontaining the single ribonucleotide residue (rA) is considered as thesubstrate (named 17DS), while the other strand as the enzyme (named17E). The catalytic core, which was highly conserved during selection,consists of a 3 bp hairpin and a 5 nt single-stranded bulge (FIG. 5).

Although ZnR17 was selected in Zn²⁺, it does not contain structuremotifs that were discovered in several Zn(II)-binding RNA molecules(Ciesiolka et al., 1995; Ciesiolka & Yarus, 1996). However, theconserved region of ZnR17 is very similar to that of the {tilde over(8)}17 deoxyribozymes selected by Santoro and Joyce using Mg²⁺ ascofactor (Santoro & Joyce, 1997). The unpaired bulge region in the{tilde over (8)}17 DNA enzyme has the sequence 5′-WCGR-3′ or 5′-WCGAA-3′(W=A or T; R=A or G). The highest activity was observed with thesequence containing 5′-TCGAA-3′. Among the Zn(II)-dependentdeoxyribozymes we obtained after reselection, 85% of them fell withinthe 5′-WCGAA-3′ regime (W=A or T). However, the sequence of the twodouble helices flanking the catalytic core is different between the{tilde over (8)}17 (FIG. 6D) and the 17E deoxyribozymes (FIG. 6F),reflecting their different designs of the selection pool. Similarsequence motif was also observed in an RNA-cleaving deoxyribozyme (namedMg5) selected by Faulhammer and Famulok using 50 mM histidine and 0.5 mMMg²⁺ as cofactors (Faulhammer & Famulok, 1997). The homologous region in31 out of the 44 sequenced clones had the sequence5′-TX₁X₂X₃AGCY₁Y₂Y₃ACGAA-3′ (SEQ ID NO:9), falling within the WCGAA-3′regime. The authors predicted a secondary structure different from the{tilde over (8)}17 or 17E motif based on chemical modification analysis.However, a structure containing a three-way junction similar to that ofthe 17E and {tilde over (8)}17 deoxyribozymes is consistent with thechemical mapping results.

Sequence Alignment and Structure Analysis of Co-DNA

The sequences of the cis-cleaving deoxyribozyme selected in the presenceof Co²⁺ are more diverse than the Zn-DNA. They can be divided into threemajor classes based on sequence similarity (FIG. 3). There is noconsensus sequence region among different classes. The secondarystructure of each sequence class of Co-DNA was predicted with DNA mfoldprogram. The minimal conserved sequence motif of class I Co-DNA includesa bulged duplex. The cleavage site is within the 13 nt single-strandedbulge. A 4 bp hairpin is also highly conserved and linked to the bulgedduplex through 3 unpaired nucleotides. The folding of the sequencesoutside of this minimal motif was highly variable and resulted instructures with a wide range of stabilization energy.

The class II Co-DNA contains a sequence region (5′-ACCCAAGAAGGGGTG-3′(SEQ ID NO:10)) that was also found in an RNA-cleaving deoxyribozyme(termed G3) selected by Geyer and Sen (1997) (FIG. 7A and 7B). Theminimal motif predicted for class II Co-DNA shows similarity to thatproposed for the G3 deoxyribozyme as well. The G3 deoxyribozyme wasbelieved to be fully active in the absence of any divalent metal ions.Copious use of divalent metal chelating agents, such as EDTA, andaccurate trace-metal analysis of the reaction solutions were used torule out the need of the G3 deoxyribozyme for contaminating levels ofdivalent metals. As mentioned earlier, the activity of class II Co-DNAwas the same in buffer alone or with added Co²⁺ or Zn²⁺, suggesting thatthis class of Co-DNA most likely contain the divalent metal-independentstructure motif.

Effect of Metal Ions on the Activity of the Cis-Cleaving Deoxyribozymes

ZnR17 and Co-18 were examined for their activity dependence onmonovalent ions and divalent metal ions other than Zn²⁺ and Co²⁺. In thepresence of 1 mM EDTA and without added Zn²⁺ ions, no cleavage wasobserved with ZnR17 even after two days, strongly suggesting thatdivalent metal ions are required for the activity of ZnR17. Although thecis-cleaving Zn-DNA was selected in the presence of 500 mM NaCl, NaClwas actually inhibitory to enzymatic activity. With 0-2 M NaCl added tothe reaction buffer (100 μM Zn²⁺, 50 mM HEPES, pH 7.0), k_(obs)decreased with increasing NaCl concentration. The deleterious effect ofNaCl was also manifested by lowered final percentage of cleavageproducts. For instance, only 50% of ZnR17 could be cleaved in thepresence of 2 M NaCl even after long incubation times, while >95% of theDNA was cleavable in the absence of extra NaCl. Contrary to the Zn-DNA,the activity of Co-18 relies on NaCl and no cleavage was observed in theabsence of NaCl. With 1 M NaCl, 8% of Co-18 molecules were cleavedwithin 5 min, while <0.2% were cleaved in the absence of extra NaCl.

Although the deoxyribozymes were selected using either zinc or cobalt ascofactor, they are also active in other transition metal ions and inPb²⁺. The cleavage efficiency of ZnR17 followed the trend ofPb²⁺>Zn²⁺>Mn²⁺˜Co²⁺˜Ca²⁺˜Cd²⁺>>Ni²⁺>Mg²⁺. It is noteworthy that thecleavage rate in Ca²⁺ was much higher than in Mg²⁺, a similar trend wasobserved with the Mg5 deoxyribozyme. The order of Co-18 activity is asfollow: Zn²⁺>Pb²⁺˜Co²⁺>Ni²⁺>Cd²⁺˜Mn²⁺>Mg²⁺˜Ca²⁺. In general, both ZnR17and Co-18 are more active in transition metal ions than inalkaline-earth metals, and higher activities were achieved with Pb²⁺,Co²⁺ and Zn²⁺. The preference of the selected deoxyribozymes for Co²⁺and Zn²⁺ reflected their selection criteria. A similar trend(Pb²⁺>Zn²⁺>Mn²⁺>Mg²⁺) was also observed with all four RNA-cleavingdeoxyribozymes selected in parallel by Breaker and Joyce using one ofthe four metal ions (Pb²⁺, Zn²⁺, Mn²⁺, Mg²⁺) as cofactor (1995). Theproposed secondary structures of the deoxyribozymes selected in Pb²⁺ andMg²⁺ have been reported (Breaker & Joyce, 1994, 1995). No structuresimilarity was observed between ZnR17 and those RNA-cleavingdeoxyribozymes.

SUMMARY

Using in vitro selection technique, several groups of RNA-cleavingdeoxyribozymes were isolated using Zn²⁺ or Co²⁺ as cofactor. No commonsequence or structural features were observed between theCo(II)-dependent and the Zn(II)-dependent deoxyribozymes, in spite ofthe chemical similarities between these two transition metal ions. Thedeoxyribozymes selected in Zn²⁺ share a common motif with the {tildeover (8)}17 and the Mg5 deoxyribozymes isolated under differentconditions, including the use of different cofactors. Both the Co-DNAand the Zn-DNA exhibited higher activity in the presence of transitionmetal ions than in alkaline earth metal ions, which are the mostcommonly adopted metal cofactors by naturally occurring ribozymes.

Example 3 Deoxyribozyme Based Biosensor for Pb²⁺ Detection

This Example describes a particle-based biosensor for the detection ofPb²⁺. The biosensor utilizes the deoxyribozyme developed in Example 2(termed 17E) combined with particle technology to allow quantitative andreal time measurements of catalytic activity. Because catalytic activityis dependent on Pb²⁺, the biosensor provides real-time, quantitative,and sensitive measurements of Pb²⁺ concentrations.

DNA/RNA Chimera Oligonucleotide Cleavable Substrate.

The oligonucleotides were purchased from Integrated DNA Technology, Inc,Coralville, Iowa. The cleavable substrate is a 44 base DNA/RNA chimeraoligonucleotide with the sequence5′-TGTCAACTCGTG-ACTCACTATrAGGAAGAGATG-TGTCAACTCGTG-3′(SEQ ID NO:85) ),in which rA represents a ribonucleotide adenosine. The cleavablesubstrate strand is derived from the substrate 17DS5′-ACTCACTATrAGGAAGAGATG-3′ (SEQ ID NO:2) (See Example 2). 17DS has beenextended on both the 3′ and 5′ ends for twelve bases, which act as“sticky end” for annealing to a complementary DNA strand on the goldparticles.

Thiol-Modified DNA Gold Particles.

Thiol-modified oligonucleotides were purchased from Integrated DNATechnology, Inc, Coralville, Iowa. A thiol-modified 12mer DNA primer,5′-SH—(CH₂)₆-CACGAGTTGACA-3′(SEQ ID NO:86) was attached to the 13 nmdiameter gold particles (produced as in Example 1) by adding the 3.6 μMthiol-modified DNA to the gold colloid. The solution was allowed to sitfor 24 hrs, and then diluted to give a final concentration of 100 mMNaCl and 25 mM Tris-acetate buffer, pH 7.2. After another 24 hrs, theDNA attached gold particles were centrifuged at 14,000 rpm for one hour.The supernatant was removed and the concentrated gold particles washedwith 100 mM NaCl buffer. After another centrifuge at 14,000 rpm, theparticles were stored in 300 mM NaCl, 25 mM Tris-acetate buffer.

Preparation of Agglutinated Gold Particles.

500 pmol cleavable substrate and 1,000 pmol trans-cleaving deoxyribozyme(17-E) were added to DNA linked gold particles (absorption of 2.2 at 522nm) to a final volume of 3 mL. The sample was buffered at pH 7.2 using25 mM Tris-acetate (TA) and 500 mM NaCl. The mixture was heated to 70°C. and allowed to cool slowly to room temperature. A dark purpleprecipitation was formed at the bottom of the tube. This is due to theaggregation of 13 nm gold particles by the DNA linker. Aftercentrifugation for one minute, the precipitation were collected andresuspended in 4 mL of 300 mM NaCl, 25 mM TA buffer pH7.2.

The trans-cleaving deoxyribozyme is required for aggregate formation.Without the deoxyribozyme, the DNA modified gold particles and cleavablesubstrate did not aggregate. A deoxyribozyme titration experiment wasperformed to determine the optimum ratio of deoxyribozyme and cleavablesubstrate. The extinction at 522 nm was shown to represent the relativeamount of separated gold particles while the extinction at 700 nm wasshown to represent the relative amount of gold particle aggregates.Hence, the ratio of extinction at 700 nm to 522 nm was used to monitorthe degree of aggregation. This procedure minimized artifacts due toconcentration differences. Aggregation increased with deoxyribozymeconcentration until the ratio of deoxyribozyme to μ reached one.

UV-Vis Extinction Spectrum of Separated and Aggregated Gold Particles

The UV-vis extinction spectrum of separated and aggregated goldparticles was determined as follows. Gold particles were prepared as inExample 1. Aggregated gold particles were prepared as in Example 3.UV-vis extinction spectra were obtained by monitoring the surfaceplasmon band of gold nanoparticles. For an non-aggregated particles, theextinction peak is 522 nm and decreases rapidly at longer wavelengths,giving the suspension a deep red color. When the particles are in theaggregated state, the extinction peak shifts to a longer wavelength andfalls off less quickly. A suspension of aggregated particles develops apurple color.

Example 4 Color Change of the Pb²⁺ Sensor in the Presence of Pb²⁺ Due toEnzymatic Substrate Cleavage

To verify that the cleavage is carried out by the deoxyribozyme 17E andusing Pb²⁺ only as a cofactor, a control experiment replacing 17E withan inactive deoxyribozyme 17E-C: 5′-CATCTCTTCCCCGAGCCGGTCGAAATAGTGAGT-3′(SEQ ID NO:87) was performed. Previous kinetics assays have shown thatthe activity of 17E was complete lost when the G•T wobble pair wasreplaced by a G•C Watson-Crick base pair. (Santoro, S. W. & Joyce, G. F.(1997); Faulhammer, D. & Famulok, M. (1997).) 17E-C is an inactiveversion of 17E by changing the important T base to a C base. Since onlyone base is changed, the structure of the substrate-17E duplex is verysimilar to the duplex formed by the substrate and 17E-C.

Aggregated particles were prepared as in Example 3. In addition,aggregated particles containing the inactive deoxyribozyme, 17E-Cinstead of the active deoxyribozyme were also prepared using the samemethod. A Pb²⁺ assay was then performed as follows. 48 μL of particlesuspension was pipetted into microcentrifuge tubes. 2 μL of a 5 μMPb²⁺standard (Pb(OAc)₂ (Aldrich, St. Louis, Mo.) in water) was added to eachtube and the tubes heated above 50° C. for 5 minutes. The meltingtemperature of the aggregated particles was previously determined bymeasuring the extinction at 260 nm. The DNA linked gold particles showeda sharp melting temperature (46° C.). Thus, heating the aggregatedparticles to a temperature higher than 50° C. resulted in the fullymelting of the aggregate.

The tubes were allowed to cool naturally to room temperature and UV-visspectra measured. When active deoxyribozyme was present, cleavage of thesubstrate was observed at 5 μM Pb²⁺. However, with inactivedeoxyribozyme, cleavage was not observed, even in the presence of 5 μMPb²⁺.

Example 5 Semi-quantitative and Quantitative DNA Pb²⁺ Assays

Semi-quantitative protocol—An assay was performed as in the previousexample. After the tubes cooled at room temperature, a 10 μL sample wasremoved and used to spot on an alumina TLC plate (Analtech, Newark,Del.). A color progression from purple (no lead present) to red (highlead concentration) was observed. Hence, the Pb²⁺ concentration in asample may be determined by comparing the color obtained with the samplewith that obtained for standard Pb²⁺ solutions or with a standard colorQuantitative protocol—After the tubes cooled at room temperature UV-visspectra were obtained using a Hewlett-Packard 8453 spectrophotometer. Toeliminate the effect of concentration, the ratio of extinction at 522 nmand 700 nm was used to monitor the degree of aggregation of particles.These two wavelengths were chosen to represent the relative amount ofaggregated and free gold particles.

Example 6 Detection Range of a DNA Pb²⁺ Assay

Agglutinated gold particles were prepared as in Example 3 except that a1 to 1 ratio of deoxyribozyme to substrate was used. A Pb²⁺ assay wasperformed as in Example 4. Pb²⁺ standards in the range 0-5 μM weretested and the results visualized on a TLC plate and by UV-visextinction spectroscopy. Pb²⁺ standards in the range from 250 nM to 1 μMwere distinguishable visually on the plate. A second assay was performedusing a 1 to 20 mixture of active and inactive deoxyribozyme. Here, theassay the detection region was from 10 μM-200 μM. Hence, by simplyvarying the amount of active deoxyribozyme, the detection range of theassay may be varied to adopt to different detection requirements. Inaddition to visual detection, results were determined quantitativelyusing the ratio of extinction at 522 nm and 700 nm.

Example 7 Cross-reactivity Characteristics of a DNA Pb²⁺ Assay

Standards containing 5 μM Pb²⁺, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ andCd²⁺ ions in water were prepared by dissolving Pb(OAc)₂, MgCl₂, MnCl₂,Col₂, NiCl₂, CuCl₂, ZnCl₂, and Cd(OAc)₂ (Aldrich, St. Louis, Mo.) inwater. A Pb²⁺ assay was performed as in Example 4 to determine thecross-reactivity of 5 μM Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and Cd²⁺.The standards containing these ions were tested along with the standardcontaining 5 μM Pb²⁺. These divalent metal ions are chosen for assaybecause they have shown relative high activities as a metal cofactor forenzymatic cleavage. At 5 μM, all seven metal ions give a blue color on aTLC plate. However, 0.5 μM Pb²⁺ developed a purple color. In addition,results were determined quantitatively using the ratio of extinction at522 nm and 700 nm. The results obtained with Mg²⁺, Co²⁺, and Zn²⁺ areshown in Table 3. Of the ions tested, Zn²⁺ and Co²⁺ showed the highestcross reactivity.

TABLE 3 Detection of Pb²⁺ in the presence of Mg²⁺, Co²⁺, and Zn²⁺ ions.Extinction Extinction Sample Name at 522 nm at 700 nm E522/E700 purewater 0.91982 0.43821 2.099 1 μM Pb²⁺ 1.0839 0.17929 6.045 1 μM Pb²⁺ + 1μM Co²⁺ 1.0844 0.1582 6.854 1 μM Pb²⁺ + 1 μM Mg²⁺ 1.0911 0.1739 6.274 1μM Pb²⁺ + 1 μM Zn²⁺ 1.1691 0.12972 9.0124

Example 8 Room Temperature DNA Pb²⁺ Assay. (Prophetic)

This example describes a room temperature DNA Pb²⁺ Assay. Thiol-modifiedoligonucleotides may be purchased from Integrated DNA Technology, Inc.,Coralville, Iowa. The thiol-modified 12mer DNA primer,5′-SH—(CH₂)₆-CACGAGTTGACA-3′(SEQ ID NO:88) is used to modify goldparticles as in Example 3. In addition, a second type to modified goldparticles are produced using the same primer sequence but with a3′-thiol linkage. Agglutinated gold particles are prepared using themethod described in Example 3 except that a 1:1 mixture of 3′-thiol and5′-thiol DNA linked particles is used.

A Pb²⁺ assay is then performed as follows. 48 μL of particle suspensionis pipetted into microcentrifuge tubes. 2 μL of a Pb²⁺ standard is addedto each tube and the tubes incubated at room temperature. The UV-visextinction spectra are then determined. A breakdown of aggregation isobserved with increasing Pb²⁺ concentration. Thus, the modified assaydoes not require the heating and cooling steps of the unmodified assay.

Example 9 Detection of Pb2+ in Leaded Paint

The DNA based sensor was used to detect Pb²⁺ in leaded paint. Leadedpaint containing various concentrations of lead was simulated by addingbasic lead carbonate 0%, 0.5%, 1%, 2%, 3%, 5% 10% by weight ofPbCO₃.Pb(OH)₂ (Aldrich, St. Louis, Mo.) to a commercial white paintcontaining a TiO₂ pigment. (Exterior white paint, Glidden, Cleveland,Ohio). The leaded paint was dissolved in 10% HOAc. After diluting thissolution 150000 times in water, a Pb²⁺ assay was performed. Results weredetermined in a modified semi-quantitative protocol. Briefly, 2 μL of 50times concentrated aggregated particles (prepared as in Example 3) wereadded to 98 μL of the diluted paint solution. This mixture was thenheated to 50° C. for 5 minutes and allowed to cool slowly to roomtemperature. A 10 μL sample was spotted onto a TLC plate. A colorpattern developed on the TLC plate showing a color transition between 2and 3% indicating that the Pb²⁺ concentrations in the original solutionsis approximately 500 nM. With the knowledge of the times of dilution,the concentration of the original soaking solution can be calculated.

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1. A method of detecting the presence of an ion in a sample, comprising:(a) forming a mixture comprising: (i) the sample; and (ii) an aggregate,wherein the aggregate comprises a plurality of complexes, wherein eachcomplex comprises (I) a nucleic acid enzyme; (II) particles; (III) afirst oligonucleotide attached to a solid surface of the particles; (IV)a second oligonucleotide attached to the solid surface of the particles;and (V) a substrate for the nucleic acid enzyme, wherein the substratehybridizes to the first oligonucleotide and the second oligonucleotide,wherein the nucleic acid enzyme depends on the ion to cause cleavage ofthe substrate and cleavage of the substrate followed by heating to 50°C. results in de-aggregation of the aggregate; and (b) detectingdeaggregation of the aggregate.
 2. The method of claim 1, wherein theion is AsO₄ ³⁻.
 3. The method of claim 1, wherein the ion is selectedfrom the group consisting of K⁺, Na⁺, Li⁺, Tl⁺, NH4⁺, Ag⁺, Mg²⁺, Ca²⁺,Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Hg²⁺, Hg₂ ²⁺, Pt²⁺, Ra²⁺, Ba²⁺, UO₂²⁺, Sr²⁺, Co³⁺, Cr³⁺, Ln³⁺, Ce⁴⁺, and Cr⁶⁺.
 4. The method of claim 1,wherein the ion comprises a member selected from the group consisting ofK (I), Na(I), Li(I), Tl(I), Ag(I), Hg(I), Mg (II), Ca (II), Mn (II),Co(II), Ni (II), Zn (II), Cd (II), Pb(II), Hg (II), Pt (II), Ra (II), Ba(II), Sr (II), Co (III), Cr (III), Ln (III), Ce (IV), Cr (VI) and U(VI).
 5. A sensor for detecting the presence of an ion in a sample,comprising: (i) an aggregate, wherein the aggregate comprises aplurality of complexes, wherein each complex comprises (I) a nucleicacid enzyme; (II) particles; (III) a first oligonucleotide attached to asolid surface of the particles; (IV) a second oligonucleotide attachedto the solid surface of the particles; and (V) a substrate for thenucleic acid enzyme, wherein the substrate hybridizes to the firstoligonucleotide and the second oligonucleotide, wherein the nucleic acidenzyme depends on the ion to cause cleavage of the substrate andcleavage of the substrate results in deaggregation of the aggregate. 6.The sensor of claim 5, wherein the particles are gold particles.
 7. Thesensor of claim 5, wherein the particles comprise at least two sets,wherein: (a) a first oligonucleotide is linked to a first set ofparticles and a second oligonucleotide is linked to a second set ofparticles; and (b) the base sequence of the first oligonucleotide isdifferent from the base sequence of the second oligonucleotide.